Localized Detection of Superoxide Generation and Monitoring of Photosensitizer Performance in Hypoxic Tumors by an In Situ Precipitating Fluorescent Probe

Type I photodynamic therapy (PDT), generating superoxide anion radicals (O₂^•-), presents a potent strategy against tumor resistance by virtue of its low oxygen dependence, ensuring efficacy in hypoxic tumors where traditional Type II PDT is limited. However, accurate O₂^•- detection with high spatiotemporal and subcellular resolution remains a critical unmet need. Herein, we report the rational design and synthesis of <b>HPQ-CF</b>_<b>3</b>, a novel fluorescent probe. Upon selective reaction with O₂^•-, <b>HPQ-CF</b>_<b>3</b>'s trifluoromethanesulfonate group departs, unmasking a hydroxyl group to generate the highly fluorescent <b>HPQ-OH</b>, a fluorophore incorporating the HPQ moiety. Crucially, the inherent low aqueous solubility of <b>HPQ-OH</b> leads to its in situ precipitation, enabling precise spatial localization of O₂^•- generation sites. <b>HPQ-CF</b>_<b>3</b> exhibits high specificity, a rapid response (<300 s), a remarkable ∼200-fold fluorescence enhancement, a low limit of detection (LOD, 0.13 μM), and excellent linearity. <b>HPQ-CF</b>_<b>3</b> demonstrated significantly superior performance over the commercial probe DHR123 for intracellular O₂^•- detection; its unique precipitation-based mechanism inherently minimizes background fluorescence while anchoring the signal at its origin, affording a substantially enhanced signal-to-noise ratio and markedly improved localization accuracy. Furthermore, <b>HPQ-CF</b>_<b>3</b> successfully monitored O₂^•- production in complex biological settings, including solid tumors. <b>HPQ-CF</b>_<b>3</b> is anticipated to be an invaluable tool for investigating O₂^•--related pathophysiology and advancing Type I photosensitizer development.